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Reengineering Cro protein functional specificity with an evolutionary code.

Branwen M Hall1, Erin E Vaughn, Adrian R Begaye

  • 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA.

Journal of Molecular Biology
|September 28, 2011
PubMed
Summary
This summary is machine-generated.

Researchers tested an evolutionary code to reengineer transcription factor specificity in Cro proteins. This code successfully switched DNA binding for some variants, confirming its potential for altering protein-DNA interactions.

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Area of Science:

  • Molecular Biology
  • Evolutionary Biology
  • Biochemistry

Background:

  • Cro proteins from lambdoid bacteriophages exhibit variable DNA target specificities, making them a model for studying transcription factor evolution.
  • An evolutionary code linking amino acid changes in Cro proteins to DNA binding specificity has been proposed based on bioinformatics.

Purpose of the Study:

  • To experimentally validate a bioinformatically derived evolutionary code relating amino acid substitutions in Cro proteins to changes in their DNA binding specificity.
  • To assess the potential for reengineering Cro protein specificity using this evolutionary code.

Main Methods:

  • Generation of all eight possible code variants of bacteriophage λ (lambda) Cro protein.
  • Electrophoretic mobility shift assays (EMSAs) to compare binding affinities of wild-type and variant Cro proteins to various DNA consensus sites.
  • Fluorescence anisotropy and bacterial one-hybrid assays to confirm specificity switches and assess binding affinity and specificity.

Main Results:

  • Most generated Cro variants showed enhanced binding to their predicted cognate DNA sites compared to the wild-type site, with some exceptions.
  • While specificity switches were observed, most engineered variants exhibited lower overall binding affinity and specificity than wild-type λ Cro.
  • Fluorescence anisotropy and bacterial one-hybrid assays confirmed specificity changes, particularly at coded amino acid positions.

Conclusions:

  • The experimental results largely support the proposed Cro evolutionary code for predicting and reengineering transcription factor specificity.
  • The study demonstrates the feasibility of altering Cro protein DNA binding specificity, although achieving high affinity and specificity simultaneously requires further refinement.
  • This work provides a foundation for understanding and manipulating protein-DNA interactions through evolutionary principles.